The proposed studies are designed to characterize the properties and behavior of a gonadotropin-responsive steroidogenesis activator peptide (SAP) in the rat ovary and testis. This cycloheximide-sensitive factor (the putative "labile protein") is comparable or identical to a similar peptide, previously isolated from the rat adrenal cortex, which activates the mitochondrial side-chain cleavage of cholesterol in a cAMP-dependent manner. To achieve that objective, the following systems will be used as appropriate: dispersed, purified normal rat Leydig cells; rat Leydig cell tumor (H-540) implants; dispersed rat luteal cells; and expressed rat preovulatory granulosa cells. The sequence of the activator peptide will be determined and a radioimmunoassay for detection and quantitation will be developed. This will permit the assessment of time and concentration-dependent changes in the intracellular content of peptide in response to gonadotropins, modulators of gonadotropin action, and inhibitors of protein translation and transcription. The testes of aging rats will also be inspected for the development of any lesions affecting this system. Details of the mechanism by which cAMP promotes a rise in the level of activator peptide will be investigated, with particular effort focused on a model invoking proteolytic generation of the functional peptide from an inactive precursor. To this end, a reticulocyte cell-free translation system and pulse-chase experiments will be employed. Additional studies are designed to assess the metabolic fate of the peptide. Insight into the mechanism by which the gonadal peptide activates cholesterol side-chain cleavage will be had by testing for various properties which might sensibly be attributed to it. These include the capacity to : promote cholesterol interaction with cytochrome P-450scc; modulate phospholipid metabolism or membrane cholesterol distribution; bind cholesterol, pregnenolone, or calcium; allosterically regulate some mitochondrial enzyme or structural protein associated with the cholesterol side-chain cleavage reaction; or facilitate the sequestration of mitochondrial calcium. The results of these investigations should markedly enhance our understanding of the way in which gonadotropins regulate a key reaction common to the steroidogenic pathways of target organs, and may well have relevance for current efforts to control reproductive function in man. In a broader context, they will expand our knowledge in what is presently a nascent field: the role of intracellular peptides as second and third messengers of endocrine control.